HPAC is a special form of affinity chromatography. Originally, affinity chromatography was performed at low pressures using media composed of beaded dextrans, celluloses, or agarose. Affinity chromatography is a form of chromatography which exploits the biological affinity of two or more molecules for one another. For example, biologically important enzymes bind to substrates with high affinity and specificity. By attaching the substrate to a suitable medium by some covalent or non-covalent linkage, the medium can then be packed into a column using aqueous buffers. When a mixture containing the enzyme of interest is applied to this column, the enzyme binds specifically to its substrate and remains in the column while most other components of the mixture have no affinity for the substrate and pass through the column un-retained. Later, the conditions can be changed to disrupt the enzyme-substrate binding and the enzyme elutes from the column in a highly purified form. The conditions which cause elution may be a change in pH, removal of some necessary cofactor, or addition of high amounts of substrate to the eluting solution. The same concept can be used to exploit the high affinity of antigens for antibodies, drugs for receptors, metals for certain proteins and nucleic acids, etc. Virtually any specific biological interaction of sufficiently high affinity can be exploited in affinity chromatography.
Closely related to affinity chromatography is a new method called Catalytic Chromatography reported by Jurado, et al., Analytical Biochemistry, Volume 282, pp. 39-45 (2000) which involves eluting enzymes from immobilized substrate columns by using the enzyme""s catalytic activity to convert the substrate to product to cause elution. Catalytic chromatography can also be performed at high pressures and would benefit from the invention described. Here, affinity chromatography will be discussed but most of what would be said about it would also apply to catalytic chromatography and other variations of the affinity chromatography technique known to those having ordinary skill in the art of affinity chromatography.
Affinity chromatography requires three essential elements: 1) a chromatography medium which is stable to the conditions of chromatography, 2) some biologically relevant molecule with affinity for the subject of separation and 3) a method for attaching the molecule to the medium. The present invention relates primarily to the third.
At low pressures (1-6 bar), beaded polysaccharides provide an adequate medium. The most common way in which biological molecules are attached to these media is by activating them using cyanogen halides such as cyanogen bromide (CNBr) using chemistry originally pioneered by Axen, et al., Nature, Volume 214, pp. 1302-1304. This reaction occurs in strongly basic NaOH (pH 11) aqueous mixtures. The site of the activation reaction is with the hydroxyl groups on the polysaccharide medium. Indeed, virtually any compound containing a hydroxyl group will react with cyanogen halides to yield reactive cyanate ester and imidocarbonate derivatives. While both cyanate esters and imidocarbonate derivatives can result from the cyanogen bromide reaction, it is the cyanate esters that are most reactive and account for chemical coupling reactions, according to Kohn and Wilchek, Analytical Biochemistry, Volume 115, pp. 375-382 (1981). These cyanate esters in turn are particularly reactive with amines and other strong nucleophiles. For example, DiRusso, et al., Journal of Chromatography, Volume 677, pp. 45-52 (1994) used this reaction to activate Sepharose (a commercially available beaded agarose) with cyanogen bromide and then to chemically couple an oligonucleotide containing a 5xe2x80x2-aminoethyl group (5xe2x80x2xe2x80x94NH2xe2x80x94CH2xe2x80x94CH2xe2x80x94(dT)18) for affinity chromatography.
The importance of the cyanogen bromide activation reaction to modern science can hardly be overstated. The vast majority of affinity chromatography that has ever been performed used this coupling chemistry and literally thousands of separations have been reported based upon this coupling chemistry.
While this low pressure affinity chromatography is a very powerful technique, it is insufficient to provide the highest chromatographic resolution. Chromatographic theory and practice has shown that the highest resolution and best performance is obtained with very small diameter beaded support media. Unfortunately, as bead diameter decreases, the operating pressures required to maintain a certain flow rate increase. Current high performance (pressure) liquid chromatography (HPLC) uses 3-5 xcexcm bead diameters and pressures as high as 350 bar. In contrast, beaded agaroses of 40-120 xcexcm are normally used at low pressures of 1-6 bar and would be destroyed at higher pressures. To resist these high pressures, silica is the most commonly used HPLC support. To mask silanols on the silica surface which irreversibly absorb many important biological molecules such as proteins, the silica surface is coated by reaction with silanes. Most HPAC uses silicas which are initially reacted with glycidyloxypropyl silanes. This reaction produces an sepoxide-silica which itself will react slowly with amino ligands. The epoxide can also be hydrolyzed in weak acids to yield a diol silica and several chemistries exist for activating these hydroxyls for amino ligand coupling. For example, Larsson, et al., Advances in Chromatography, Volume 21, pp. 41.84 (1983) and Larsson, Methods in Enzymology, Volume 104, pp. 212-223 (1984) and Ernst-Cabrera and Wilchek, Journal of Chromatography, Volume 397, pp.187-196 (1987) have reported several such chemistries. For various reasons, none of these chemistries has been widely used. Some chemistries have reacted only slowly or in poor yield. Another inhibiting factor is that most of affinity chromatography has been performed with the cyanogen bromide activated supports and adapting each of these methods to a new chemistry, even if performance could be improved, would require a great deal of effort.
While silica is very pressure stable, it is unstable at pH values above pH 8 and is dissolved by strongly alkaline solutions such as those used in classical CNBr activation. An alternative to the use of NaOH has been provided by Kohn and Wilchek, Biochemical and Biophysical Research Communications, Volume 107, pp. 878-884 (1982). Using 60% acetone and the organic base triethylamine (TEA) in place of NaOH, they were able to activate agarose and other polysaccharide based support media with cyanogen bromide. While this reaction is somewhat safer to perform than the traditional CNBr chemistry, it still uses a strong organic base (TEA) which would be detrimental if used with silica. To perform other chemistry unrelated to this patent, Jarrett, Journal of Chromatography, Volume 405, pp. 179-189 (1987) had found that silica is not dissolved by organic bases such as pyridine if anhydrous conditions are maintained. This suggested to us that we may be successful in using a reaction similar to that of Kohn and Wilchek, Biochemical and Biophysical Research Communications, Volume 107, pp. 878-884 (1982) with silica except using strictly anhydrous conditions rather than aqueous acetone. This has now been demonstrated and provides the basis of the invention.
It is the object of the present invention to provide a medium for HPAC which is pressure stable, which shows improved resolution over current media, and uses the familiar CNBr chemistry.
It is a further object of this invention to provide a method for producing such a medium.
An examination of the following disclosure will allow a person having ordinary skill in the art to discern other objects and advantages of the present invention.
The present invention provides for a method to prepare a pressure stable and pH stable medium for use in high pressure (performance) affinity chromatography. The method comprises the steps of treating hydroxyalkyl-silica with cyanogen halides or other cyanogen transfer reagents in the presence of an organic base in anhydrous solvents at temperatures in the range of from about xe2x88x9215xc2x0 C. to about 20xc2x0 C. for a period of time in the range of from about 1 minute to about 5 minutes, and washing the resulting medium in anhydrous solvent.
In a preferred embodiment of the invention, the hydroxyalkyl-silica is glycidioloxypropyl-silica. In another preferred embodiment of the invention, the cyanogen transfer reagent is cyanogen bromide. In a further preferred embodiment of the invention, the organic base is triethylamine. In yet another preferred embodiment of the invention, the anhydrous solvent is selected from the group consisting of acetone, N,N-dimethylformamide, and 2-propanol. In a further preferred embodiment of the invention, the temperature is xe2x88x9215xc2x0 C. and the period is 3 minutes. In another preferred embodiment of the invention, the temperature is 20xc2x0 C. and the period is about 5 minutes.
In order to provide a better understanding of the present invention, the following Figures, along with the following Examples, are given by way of illustration to show certain specific details thereof.